Glycine-substituted thieno{2,3-d}pyrimidines with combined lh and fsh agonistic activity

ABSTRACT

The present invention resides in glycine substituted thieno[2,3-d]pyrimidine derivatives according to general formula I,  
                 
 
     or a pharmaceutically acceptable salt thereof, wherein  
     X is O or H,H  
     A is S, NH, N(R 6 ), O or a bond;  
     R 1  is (1-4C)alkyl, (2-4C)alkenyl, phenyl or (2-5C)heteroaryl, the phenyl or heteroaryl ring optionally being substituted with one or more of the group of substituents: hydroxy, halogen, nitro, trifluoromethyl, cyano, amino or (1-4C)(di)alkylamino and  
     R 2  is H, (1-4C)alkyl, (1-4C)alkoxy(2-4C)alkyl or hydroxy(2-4C)alkyl;  
     R 3  and R 4  can be independently selected from H and hydroxy(1-4C)alkyl;  
     R 5  is H or (1-4C)alkyl;  
     R 6  can be selected from the same groups as described for R 1 .  
     The compounds of the invention have LH as well as FSH receptor activating activity and can be used in fertility regulating therapies.

[0001] The invention relates to compounds having glycoprotein hormoneagonistic activity, in particular to compounds having both LuteinizingHormone (LH) and Follicle Stimulating Hormone (FSH) agonistic activity.The invention furthermore relates to pharmaceutical compositionscontaining the same as well as to the use of these compounds in medicaltherapy, particularly for use as a control of fertility.

[0002] Gonadotropins serve important functions in a variety of bodilyfunctions including metabolism, temperature regulation and thereproductive process. The hypophyseal gonadotropins FSH and LH forexample play a pivotal role in the stimulation of follicle developmentand maturation whereas LH is involved in induction of the ovulatoryprocess (Sharp, R. M. Clin. Endocrinol 33:787-807, 1990; Dorrington andArmstrong, Recent Prog. Horm. Res 35: 301-342, 1979; Levy et al, HumanReproduction 15:2258-2265, 2000).

[0003] Currently, LH is applied clinically, in combination with FSH, forovarian stimulation i.e. ovarian hyperstimulation for in vitrofertilisation (IVF) and induction of ovulation in infertile anovulatorywomen (Insler, V., Int. J. Fertility 33:85-97, 1988, Navot andRosenwaks, J. Vitro Fert. Embryo Transfer 5:3-13, 1988), as well as formale hypogonadism and male infertility.

[0004] Gonadotropins act on specific gonadal cell types to initiateovarian and testicular differentiation, and steroidogenesis. The actionsof these pituitary and placental hormones are mediated by specificplasma membrane receptors that are members of the large family ofG-protein coupled receptors. They consist of a single polypeptide withseven transmembrane domains and are able to interact with the Gsprotein, leading to the activation of adenyl cyclase.

[0005] Gonadotropins destined for therapeutic purposes can be isolatedfrom human urine sources and are of low purity (Morse et al, Amer. J.Reproduct. Immunol. and Microbiology 17:143, 1988). Alternatively, theycan be prepared as recombinant gonadotropins. In addition to theseproteins, gonadotropin receptors can be activated or deactivated bysynthetic low molecular weight compounds. Bicyclic heteroaromaticcompounds have been described in WO 00/61586. By in vitro and in vivoexperiments they are shown to be useful as LH agonists.

[0006] In normal females the release of pituitary LH and FSH ischaracterized by a mid-cycle surge which precedes the ovulation.Ovulation is characterized by three distinct physiological phenomenai.e. oocyte maturation, follicular rupture and luteinization. While therole of the LH-surge in the in vivo induction of these phenomena isundisputed, the role of the FSH-surge is less clear. However, it hasbeen shown recently that FSH induces oocyte maturation in vitro byinducing cumulus cells to produce a factor that positively overcomeshypoxanthine induced meiotic arrest (Lu et al, Mol. Cell. Endocrinol.164:191-196, 2000). This factor is thought to be a meiosis activatingsterol (MAS).

[0007] The present invention provides low molecular weight compoundsthat show LH activity. In addition to LH activity unexpectedly they alsohave FSH activity. In general these compounds arethieno[2,3-d]pyrimidines which at the 4-position of the pyrimidine ringare substituted by a phenyl group which in turn is substituted at themeta position.

[0008] This substituent comprises a three-atom spacer (NH—C(O)—CH₂),which is further functionalized with a substituted amino group.Generally the compounds have FSH agonistic activity in varying degreesbut typically less than the LH agonistic activity.

[0009] The present invention resides in glycine substitutedthieno[2,3-d]pyrimidine derivatives according to general formula I,

[0010] or a pharmaceutically acceptable salt thereof, wherein

[0011] X is O or H,H

[0012] A is S, NH, N(R⁶), O or a bond;

[0013] R¹ is (1-4C)alkyl, (2-4C)alkenyl, phenyl or (2-5C)heteroaryl, thephenyl or heteroaryl ring optionally being substituted with one or moreof the group of substituents: hydroxy, halogen, nitro, trifluoromethyl,cyano, amino or (1-4C)(di)alkylamino and

[0014] R² is H, (1-4C)alkyl, (1-4C)alkoxy(2-4C)alkyl orhydroxy(2-4C)alkyl;

[0015] R³ and R⁴ can be independently selected from H, (1-4C)alkyl andhydroxy(1-4C)alkyl;

[0016] R⁵ is H or (1-4C)alkyl.

[0017] R⁶ can be selected from the same groups as described for R¹.

[0018] The term (1-4C)alkyl as used in the definition of formula I meansa branched or unbranched alkyl group having 1-4 carbon atoms, beingmethyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl.

[0019] The term (2-4C)alkenyl as used in the definition of formula Imeans a branched or unbranched alkenyl group having 2-4 carbon atoms,being vinyl, 1-propenyl, 2-propenyl, 1-methyl-vinyl, 1-butenyl,2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl,1-methyl-1-propenyl, 1-ethyl-vinyl.

[0020] The term (1-4C)alkoxy(1-4C)alkyl means an alkyl group having 1-4carbon atoms, attached via an oxygen atom to another alkyl group having1-4 carbon atoms, the alkyl moieties having the same meaning aspreviously defined.

[0021] The term (1-4C)alkoxy(2-4C)alkyl means an alkyl group having 1-4carbon atoms, attached via an oxygen atom to another alkyl group having2-4 carbon atoms, the alkyl moieties having the same meaning aspreviously defined.

[0022] The term hydroxy(1-4C)alkyl means an hydroxyl group attached toan alkyl group having 1-4 carbon atoms, the alkyl moiety having the samemeaning as previously defined.

[0023] The term hydroxy(2-4C)alkyl means an hydroxyl group attached toan alkyl group having 2-4 carbon atoms, the alkyl moiety having the samemeaning as previously defined.

[0024] The term (1-4C)(di)alkylamino means one or two alkyl groupshaving 1-4 carbon atoms as previously defined, attached to a nitrogenatom.

[0025] The term (2-5C)heteroaryl means an, optionally substituted,aromatic group having 2-5 carbon atoms, at least including oneheteroatom selected from N, O and/or S, like imidazolyl, thienyl, furylor pyridyl.

[0026] The term halogen means fluorine, chlorine, bromine or iodine.

[0027] It has been shown that compounds of the above mentioned formula Ishow agonistic LH and FSH activity. In an in vitro bioassay using CHOcells stably transfected with the human LH or FSH receptor,respectively, the EC₅₀ with regard to the LH receptor was found to beless than 5.10⁻⁸ M whereas with regard to the FSH receptor the EC₅₀ wasless than 10⁻⁵M. Typically the FSH activity ranges from an activity ofabout 0.1% of the LH agonist stimulation to about 10% of the LH agoniststimulation.

[0028] The invention further resides in a pharmaceutical compositioncomprising a thieno[2,3-d]pyrimidine derivative compound or saltsthereof having the general formula I.

[0029] Thus, the compounds according to the invention can be used intherapy. A further aspect of the invention resides in the use of athieno[2,3-d]pyrimidine compound having the general formula I for themanufacture of a medicament for the control of fertility, morepreferably induction of ovulation. The present compounds are used toactivate both the LH and FSH receptors. The compound of the presentinvention can be used therefore in a method to treat females withfertility problems.

[0030] The thieno[2,3-d]pyrimidine compounds of this invention maypossess one or more chiral carbon atoms. The compounds may therefore beobtained as chirally pure compounds or as a mixture of diastereomersand/or enantiomers. Methods for obtaining the chirally pure compoundsare well known in the art, e.g. crystallization or chromatography.

[0031] For therapeutic use, salts of the compounds of formula I arethose wherein the counterion is pharmaceutically acceptable. However,acid addition salts of bases according to formula I, may also find use,for example, in the preparation or purification of a pharmaceuticallyacceptable compound. All salts, whether pharmaceutically acceptable ornot, are included within the ambit of the present invention.

[0032] Examples of acid addition salts include those derived frommineral acids such as hydrochloric acid, phosphoric acid, sulphuricacid, preferably hydrochloric acid, and organic acids like citric acid,tartaric acid, acetic acid, lactic acid, maleic acid, malonic acid,fumaric acid, glycolic acid, succinic acid, and the like.

[0033] Suitable administration routes for the compounds of formula I orpharmaceutically acceptable salts thereof, also referred to herein asthe active ingredient are intramuscular injections, subcutaneousinjections, intravenous injections or intraperitoneal injections, oraland intranasal administration. Preferably, the compounds may beadministered orally. The exact dose and regimen of administration of theactive ingredient, or a pharmaceutical composition thereof, willnecessarily be dependent upon the therapeutic effect to be achieved(treatment of infertility; contraception), and may vary with theparticular compound, the route of administration, and the age andcondition of the individual subject to whom the medicament is to beadministered.

[0034] In general, parenteral administration requires lower dosages thanother methods of administration which are more dependent uponadsorption. However, a dosage for humans preferably contains 0.0001-25mg per kg body weight. The desired dose may be presented as one dose oras multiple subdoses administered at appropriate intervals throughoutthe day. In case of female recipients, doses may be administered atappropriate daily intervals throughout the menstrual cycle forfollicular support or as a single dose for ovulation induction. Thedosage as well as the regimen of administration may differ between afemale and a male recipient.

[0035] In case of in vitro or ex vivo applications, like in IVFapplications, the compounds of the inventions are to be used in theincubation media in a concentration of approximately 0.01-5 μg/ml.

[0036] The present invention thus also relates to pharmaceuticalcompositions comprising a thieno[2,3-d]pyrimidine compound according toformula I in admixture with pharmaceutically acceptable auxiliaries, andoptionally other therapeutic agents. The auxiliaries must be“acceptable” in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipients thereof.

[0037] Pharmaceutical compositions include those suitable for oral,rectal nasal, topical (including transdermal, buccal and sublingual),vaginal or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The compositions may beprepared by any method well known in the art of pharmacy, for example,using methods such as those described in Gennaro et al., Remington'sPharmaceutical Sciences (18 th ed., Mack Publishing company, 1990, seeespecially Part 8: Pharmaceutical Preparations and Their Manufacture).

[0038] Such methods include the step of bringing in association theactive ingredient with any auxiliary agent. The auxiliary agent(s), alsonamed accessory ingredients, include those conventional in the art(Gennaro, supra), such as, fillers, binders, diluents, disintegrants,lubricants, colorants, flavoring agents and wetting agents.

[0039] Pharmaceutical compositions suitable for oral administration maybe presented as discrete dosage units such as pills, tablets orcapsules, or as a powder or granules, or as a solution or suspension.The active ingredient may also be presented as a bolus or paste.

[0040] The compositions can further be processed into a suppository orenema for rectal administration.

[0041] For parenteral administration, suitable compositions includeaqueous and non-aqueous sterile injection. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedvials and ampoules, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of sterile liquid carrier, forexample, water prior to use.

[0042] Compositions, or formulations, suitable for administration bynasal inhalation include fine dusts or mists which may be generated bymeans of metered dose pressurized aerosols, nebulisers or insufflators.

[0043] The thieno[2,3-d]pyrimidine compounds of the invention can alsobe administered in the form of implantable pharmaceutical devices,consisting of a core of active material, encased by a releaserate-regulating membrane. Such implants are to be applied subcutaneouslyor locally, and will release the active ingredient at an approximatelyconstant rate over relatively large periods of time, for instance fromweeks to years. Methods for the preparation of implantablepharmaceutical devices as such are known in the art, for example asdescribed in European Patent 0,303,306 (AKZO N.V.).

[0044] Thus, the compounds according to the present invention can beused for the same clinical purposes as the native LH, with the advantagethat they possess FSH activity, display altered stability properties andcan be administered differently.

[0045] The compounds of the present invention, represented by formula(I) can generally be prepared by nucleophilic substitution of compoundsof general formula (II) wherein Q=Cl or Br with amines of generalformula (III) in an appropriate solvent such as N,N-dimethylformamide orTHF at room temperature in the presence of a tertiary base such asN,N-diisopropylethylamine. Many amines of general formula (E) arecommercially available.

[0046] Derivatives of formula (II) wherein Q=Cl or Br can be prepared byregioselective acylation of meta aniline derivatives of formula (V-a)with acyl chlorides of type (IV), wherein Q=Cl or Br in the presence ofa tertiary base such as N,N-diisopropylethylamine in a suitable solventsuch as dichloromethane or THF.

[0047] Compounds of formula (V) are accessible by art-known reduction ofthe nitro function in derivatives of formula (VI), using an appropriatereducing agent such as hydrogen in the presence of a metal (Pd/Pt)catalyst. Related reductions have been described in: P. M. Carabateas,P. R. Brundage, K. O. Gelotte, M. D. Gruett, R. R. Lorenz, J.Heterocycl. Chem. 21, 1849 (1984). Alternatively, the reduction can beeffected with tin(II) chloride in a protic solvent such as ethanol inthe presence of hydrochloric acid at elevated temperature (J. Heilbron,J. Chem. Soc, 1279 (1940)).

[0048] Thienopyrimidines of general formula (VI) are accessible bycondensation of carboxylic acids (VIE) with tert-butyl amine under theinfluence of a coupling agent such asO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU) or bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP)and a tertiary base, e.g. N,N-diisopropylethylamine.

[0049] Saponification of the corresponding ethyl esters (VIII) tocarboxylic acids (VII) takes place in the presence of a base such aslithium hydroxide, potassium hydroxide or sodium hydroxide in aqueousdioxane at elevated temperature (80° C. to reflux).

[0050] Bicyclic systems of general formula (VIII) are formed bysubstitution of chlorides of formula (X) with ethyl mercaptoacetateunder the agency of N,N-diisopropylethylamine, followed bybase-catalyzed ring-closure of the intermediate thioethers (IX). Thistype of thieno[2,3-d]pyrimidine ring formations has been described in:S. A. Abdel-Hady, M. A. Badawy, Y. A. Ibrahim, Sulfur Left. 9, 101(1989) and S. Tumkevicius, Liebigs Ann., 1703 (1995).

[0051] Suitable conditions for the cyclization reaction are sodiumethoxide in ethanol or N,N-diisopropylethylamine in toluene/ethanol(1/1, v/v) at reflux temperature.

[0052] Compounds of formula (X) can be synthesized following literatureprocedures as described for example by A. A. Santilli, D. H. Kim and S.V. Wanser, J. Heterocycl. Chem. 1,445, 1971. In a typical experiment, anamide of general structure (XI) is treated with POCl₃ at elevatedtemperature (80° C. to reflux). The addition of an appropriate solvent,e.g. dioxane, and/or the addition of either PCl₅ or N,N-dimethylanilineto the reaction mixture may result in shorter reaction times and higheryields of chlorides (X).

[0053] A general route towards lactams of formula (XI) comprisescondensation of ethyl cyanoacetate with 3-nitro-benzaldehyde andcompounds (XII), which may be isothiourea (XII-a), isourea (XII-b),monosubstituted guanidines (XII-c), disubstituted guanidines (XII-d) oramidines (XII-e).

[0054] In a typical experiment, ethyl cyanoacetate, 3-nitrobenzaldehydeand derivative (XII) are suspended in an appropriate solvent, e.g.ethanol, methanol, N,N-dimethylformamide, N-methylpyrrolidinone,tetrahydrofuran or pyridine and a base such as potassium carbonate,sodium acetate, sodium methoxide or sodium ethoxide is added. Reactiontakes place at elevated temperature (70° C. to reflux). Afterfiltration, residues are taken up in water and acidified (pH 2) afterwhich products (XI) precipitate (S. Kambe, K. Saito and H. Kishi,Synthesis, 287 (1979); A. M. Abd-Elfattah, S. M. Hussain and A. M.El-Reedy, Tetrahedron 39, 3197 (1983); S. M. Hussain, A. A. El-Barbaryand S. A. Mansour, J. Heterocycl. Chem. 22, 169 (1985)).

[0055] Alternatively, the compounds of the present invention whereinA=N, represented by formula (I-a), can be prepared from sulfoxidederivatives of general formula (XI) via nucleophilic substitution withamine nucleophiles of general structure (XIV). The reaction is typicallyconducted at elevated temperature in the presence of a tertiary basesuch as N,N-diisopropylethylamine in an appropriate solvent such as1,4-dioxane.

[0056] Similarly, compounds of the present invention wherein A=0,represented by formula (I-b), can be prepared from sulfoxide derivativesof general formula (XIII) via nucleophilic substitution with alkoxidenucleophiles of general structure (XV). The reaction is carried out inthe presence of potassium tert-butoxide with an excess of alcohol R¹—OH.

[0057] The sulfoxide derivatives of general formula (XIII) areaccessible by oxidation of compounds of general formula (I), whereinR¹=Me and A=S, represented by formula (I-c). The oxidation is effectedby 3-chloroperbenzoic acid in trifluoroacetic acid. The acidic nature ofthe solvent prevents oxidation of the 5-amino group to the corresponding5-nitroso derivative.

[0058] Methods to determine receptor binding as well as in vitro and invivo assays to determine biological activity of gonadotropins are wellknown. In general, expressed receptor is contacted with the compound tobe tested and binding or stimulation or inhibition of a functionalresponse is measured.

[0059] To measure a functional response isolated DNA encoding the LH orthe FSH receptor gene, preferably the human receptor, is expressed insuitable host cells. Such a cell might be the Chinese Hamster Ovarycell, but other cells are also suitable. Preferably the cells are ofmammalian origin (Jia et al, Mol.Endocrin., 5:759-776, 1991.

[0060] Methods to construct recombinant LH or FSH expressing cell linesare well known in the art (Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, latest edition). Expression of receptor is attained byexpression of the DNA encoding the desired protein. Techniques for sitedirected mutagenesis, ligation of additional sequences, PCR, andconstruction of suitable expression systems are all, by now, well knownin the art. Portions or all of the DNA encoding the desired protein canbe constructed synthetically using standard solid phase techniques,preferably to include restriction sites for ease of ligation. Suitablecontrol elements for transcription and translation of the includedcoding sequence can be provided to the DNA coding sequences. As is wellknown, expression systems are now available which are compatible with awide variety of hosts, including prokaryotic hosts such as bacteria andeukaryotic hosts such as yeast, plant cells, insect cells, mammaliancells, avian cells and the like.

[0061] Cells expressing the receptor are then contacted with the testcompound to observe binding, or stimulation or inhibition of afunctional response.

[0062] Alternatively isolated cell membranes containing the expressedreceptor may be used to measure binding of compound.

[0063] For measurement of binding radioactively or fluorescently labeledcompounds may be used. As reference compound human recombinant LH or FSHcan be used. In the alternative also competition binding assays can beperformed.

[0064] Another assay involves screening for LH or FSH receptor agonistcompounds by determining stimulation of receptor mediated cAMPaccumulation. Thus, such a method involves expression of the receptor onthe cell surface of a host cell and exposing the cell to the testcompound. The amount of cAMP is than measured. The level of cAMP will bereduced or increased, depending on the inhibitory or stimulating effectof the test compound upon binding to the receptor.

[0065] In addition to direct measurement of e.g. cAMP levels in theexposed cell, cells can be used which in addition to transfection withreceptor encoding DNA are also transfected with a second DNA encoding areporter gene the expression of which responds to the level of cAMP.Such reporter genes might be cAMP inducible or might be constructed insuch a way that they are connected to novel cAMP responsive elements. Ingeneral, reporter gene expression might be controlled by any responseelement reacting to changing levels of cAMP. Suitable reporter genes aree.g. LacZ, alkaline phosphatase, firefly luciferase and greenfluorescence protein. The principles of such transactivation assays arewell known in the art and are described e.g. in Stratowa, Ch, Himmler, Aand Czernilofsky, A. P. (1995) Curr.Opin.Biotechnol.6:574.

[0066] For selecting active compounds on the LH or FSH receptor, testingat 10⁻⁵ M must result in an activity of more than 20% of the maximalactivity when LH or FSH is used as a reference. Another criterion mightbe the EC₅₀ value, which must be <10⁻⁵ M, preferably <10⁻⁷ M.

[0067] The skilled artisan will recognize that desirable EC₅₀ values aredependent on the compound tested. For example, a compound with an EC₅₀,which is less than 10-5 M is generally, considered a candidate for drugselection. Preferably this value is lower than 10⁻⁷ M. However, acompound which has a higher EC₅₀, but is selective for the particularreceptor, may be even a better candidate.

[0068] Screening for LH receptor agonistic compounds can also beperformed by using a mouse Leydig cell bioassay (Van Damme, M.,Robersen, D. and Diczfalusy, E. (1974). Acta Endocrinol. 77: 655-671Mannaerts, B., Kloosterboer, H. and Schuurs, A. (1987).Neuroendocrinology of reproduction. R. Rolland et al. Eds., ElsevierScience Publishers B.V., 49-58). In this assay, stimulation of LHreceptor mediated testosterone production can be measured in Leydigcells isolated from male mice.

[0069] FSH agonistic activity of compounds can also be determined in anex vivo model using cultured mouse follicles according to Nayudu, P. andOsborn, S. (1992, J. Reproduction and Fertility 95:349-362). Therefore,mouse ovarian follicles are isolated and cultured in the presence of FSHagonistic compounds to induce follicular growth. Measurements offollicular diameter and estradiol in the culture medium are indicativefor follicular growth.

[0070] To measure LH in vivo activity of compounds, ovulation inductionin immature mice can be studied. In this assay immature female mice areprimed with urinary FSH and approximately 48 hours later treated with aLH agonistic compound. The animals are killed after LH agonist treatmentand the number of ova in the oviduct is microscopically assessed.

[0071] To measure FSH in vivo activity of compounds immature female ratsare treated at 0, 8, 24 and 32 hours with a FSH agonistic compound toinduce follicular growth. At 52 hours after the start of the experimentthe animals are injected with hCG to induce ovulation. The animals arekilled 72 hours after the start of the experiment and the number of ovain the oviduct is microscopically assessed. In addition ovarian weightis determined.

[0072] The compounds of the present invention can be applied clinicallyin those regimens where now LH or hCG is used. These include LHsubstitution among subjects with hypogonadal hypogonadism either male orfemale, midcycle administration to induce ovulation (ovulation induction(OI) or controlled hyperstimulation (COH) or stimulation of the corpusluteum.

[0073] The following examples are illustrative for the invention andshould in no way be interpreted as limiting the scope of the invention.

EXAMPLES Example 1

[0074] tert-Butyl5-amino-2-methylthio-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0075] (a). 5-Cyano-4-(3-nitrophenyl)-2-methylthio-6-hydroxy-pyrimidine

[0076] A mixture of S-methylisothiourea sulfate (69.0 g),3-nitrobenzaldehyde (75.0 g), ethyl cyanoacetate (56.0 ml) and potassiumcarbonate (72.5 g) in abs. EtOH (1500 ml) was stirred at 60° C. for 16h. The reaction mixture was cooled to 0° C. in an ice bath. Theresulting precipitate was filtered off, washed with abs. EtOH anddissolved in hot water (100° C.). The solution was cooled to roomtemperature, acidified with 2N HCl to pH 2 and cooled to 0° C. in an icebath. The resulting precipitate was filtered off and washed with icewater. Residual water in the precipitate was removed by coevaporationwith 1,4-dioxane.

[0077] Yield: 54.0 g.

[0078] MS-ESI: [M+H]⁺=289.0

[0079] TLC: R_(f)=0.3, silica gel, DCM/MeOH=9/1 (v/v).

[0080] (b). 6-Chloro-5-cyano-4-(3-nitrophenyl)-2-methylthio-pyrimidine

[0081] POCl₃ (100 ml) was added to a stirred solution of5-cyano-4-(3-nitrophenyl)-2-methylthio-6-hydroxy-pyrimidine (example1(a), 25.0 g) in dry 1,4-dioxane (300 ml). After 3 h at 90° C., themixture was cooled to room temperature and concentrated under reducedpressure. The residue was dissolved in 1,4-dioxane (100 ml) and theresulting solution was cooled to 0° C. Ice water was cautiously added.The resulting precipitate was filtered off and washed with water.Residual water in the precipitate was removed by coevaporation with1,4-dioxane.

[0082] Yield: 26.0 g.

[0083] MS-ESI: [M+H]⁺=307.0

[0084] TLC: R_(f)=0.5, silica gel, heptane/EtOAc=3/2 (v/v).

[0085] (c). Ethyl5-cyano-4-(3-nitrophenyl)-2-methylthio-6-(ethoxycarbonylmethylthio)-pyrimidine

[0086] DIPEA (15.7 ml) was added to a stirred solution of ethyl2-mercaptoacetate (9.3 ml) and6-chloro-5-cyano-4-(3-nitrophenyl)-2-methylthio-pyrimidine (example1(b), 26.0 g) in a mixture of EtOH (250 ml) and DCM (250 ml). After 1 hat room temperature, 0.1N aq. HCl (500 ml) was added to the mixturewhich was then extracted with DCM (3×500 ml), dried (MgSO₄) andconcentrated under reduced pressure.

[0087] Yield: 28.0 g

[0088] MS-ESI: [M+H]⁺=391.4

[0089] TLC: R_(f)=0.5, silica gel, heptane/EtOAc=3/2 (v/v).

[0090] (d). Ethyl5-amino-4-(3-nitrophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate

[0091] A mixture of ethyl5-cyano-4-(3-nitrophenyl)-2-methylthio-6-(ethoxycarbonylmethylthio)-pyrimidine(example 1(c), 28.0 g) and DIPEA (30 ml) in a mixture of toluene (150ml) and EtOH (150 ml) was stirred at reflux temperature (100° C.) for 16h. The mixture was then cooled to room temperature and concentratedunder reduced pressure. Residual DIPEA was removed by coevaporation withtoluene.

[0092] Yield: 28.0 g

[0093] MS-ESI: [M+H]⁺=391.4

[0094] TLC: R_(f)=0.6, silica gel, heptane/EtOAc=3/2 (v/v).

[0095] (e). Ethyl5-amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate

[0096] EtOH (400 ml) was added to a mixture of ethyl5-amino-4-(3-nitrophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate(example 1(d), 28.0 g), concentrated aq. HCl (15 ml) and tin (II)chloride (41.0 g) in 1,4-dioxane (400 ml). The mixture was stirred at90° C. for 16 h. The mixture was then cooled to room temperature andconcentrated under reduced pressure. The residue was suspended in EtOAc(1000 ml). 4N aq. NaOH was added to obtain a pH of 10-11. The mixturewas vigourously stirred and the organic layer was separated, dried(MgSO₄) and concentrated under reduced pressure.

[0097] Yield: 21.0 g

[0098] MS-ESI: [M+H]⁺=361.0

[0099] TLC: R=0.6, silica gel, heptane/EtOAc=3/2 (v/v).

[0100] (f).5-Amino-4(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylicacid

[0101] Potassium hydroxide (32.4 g) was added to a solution of ethyl5-amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate(example 1(e), 21.0 g) in a mixture of 1,4-dioxane (300 ml) and water(100 ml). After 16 h at 90° C., the mixture was cooled to 10° C. and 2Naq. citric acid (300 ml) was added under vigourous stirring. Theresulting precipitate was filtered off, washed with water (180 ml) anddried in vacuo.

[0102] Yield: 14.0 g

[0103] MS-ESI: [M+H]⁺=333.0

[0104] TLC: R_(f)=0.5, silica gel, DCM/MeOH=9/1 (v/v).

[0105] (g). tert-Butyl5-amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide

[0106] TBTU (16.1 g) was added to a solution of5-amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylicacid (example 1(f), 14.0 g), DIPEA (17.4 ml) and tert-butylamine (7.3 g)in DCM/DMF (1/1, v/v, 250 ml). After 3 h at room temperature, themixture was diluted with DCM (50 ml), washed with sat. aq. NaHCO₃ (3×100ml), 0.1 N aq. HCl (100 ml) and water (100 ml). The organic layer wasconcentrated under reduced pressure. The crude product was purified bycrystallisation from warm abs.

[0107] EtOH (300 ml).

[0108] Yield: 10.5 g

[0109] MS-ESI: [M+H]⁺=388.2

[0110] HPLC:R_(t)=30.72 min, Luna C-18(2), 5 μm, 250×2.0 mm, detectionUV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentwater/ACN/MeOH=90/9.5/0.5 to 0/95/5, run time=50 min.

[0111] (h). tert-Butyl5-amino-2-methylthio-4-(3-(2-bromoacetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0112] Bromoacetyl chloride (615 mg) was added to a solution oftert-butyl5-amino-2-methylthio-4-(3-aminophenyl)-thieno[2,3-d]-pyrimidine-6-carboxamide(example 1(g), 1.08 g) and DIPEA (2.43 ml) in dry DCM (20 ml). After 3 hat room temperature, the mixture was diluted with DCM, washed with sat.aq. NaHCO₃, dried (MgSO₄) and concentrated under reduced pressure. Thecrude product was purified by chromatography on silica gel usingheptane/EtOAc=3/2 (v/v) as eluent.

[0113] Yield: 910 mg

[0114] MS-ESI: [M+H]+l=510.2

[0115] TLC: R_(f)=0.3, silica gel, heptane/EtOAc=3/2 (v/v).

[0116] (i). tert-Butyl5-amino-2-methylthio-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0117] N-methyl-2-amino-ethanol (250 mg) was added to a solution oftert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 250 mg) in DCM (5 ml). After 16 h at room temperature,the mixture was diluted with DCM (50 ml), washed with sat. aq. NaHCO₃,dried (MgSO₄) and concentrated under reduced pressure. The crude productwas purified by HPLC using a Luna C-18 column with the followinggradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min.The title compound was then lyophilized from a mixture of 1,4-dioxane,0.1% aq. TFA and water.

[0118] Yield: 112 mg (TFA-salt)

[0119] MS-ESI: [M+H]⁺=503.2

[0120] HPLC: R_(t)=11.45 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 ml.

Example 2

[0121] tert-Butyl5-amino-2-methylthio-4-(3-((N-(1-hydroxy-2-methyl-prop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0122] 2-amino-2-methyl-propanol (250 mg) was added to a solution oftert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 250 mg) in DCM (5 ml). After 16 h at room temperature,the mixture was diluted with DCM (50 ml), washed with sat. aq. NaHCO₃,dried (MgSO₄) and concentrated under reduced pressure. The crude productwas purified by HPLC using a Luna C-18 column with the followinggradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min.The title compound was then lyophilized from a mixture of 1,4-dioxane,0.1% aq. TFA and water.

[0123] Yield: 67 mg (TFA-salt)

[0124] MS-ESI: [M+H]⁺=517.2

[0125] HPLC: R_(t)=12.67 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 3

[0126] tert-Butyl5-amino-2-methylthio-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0127] Glycine methyl ester hydrochloride (200 mg) was added to asolution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 250 mg) and N,N-diisopropylethylamine (0.20 ml) in DCM (5ml). After 16 h at room temperature, the mixture was diluted with DCM(50 ml), washed with sat. aq. NaHCO₃, dried (MgSO₄) and concentratedunder reduced pressure. The crude product was purified by TALC using aLuna C-18 column with the following gradient: 0.1% aq. TFA+10% aq.ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compound was thenlyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0128] Yield: 133 mg (TFA-salt)

[0129] MS-ESI: [M+H]⁺=517.2

[0130] HPLC: R_(t)=11.87 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 4

[0131] tert-Butyl5-amino-2-methylthio-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0132] N-(2-methoxyethyl)-ethylamine (266 mg) was added to a solution oftert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 200 mg) in DCM (5 ml). After 16 h at room temperature,the mixture was diluted with DCM (50 ml), washed with sat. aq. NaHCO₃,dried (MgSO₄) and concentrated under reduced pressure. The crude productwas purified by HPLC using a Luna C-18 column with the followinggradient: 0.1% aq. TFA+L0% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min.The title compound was then lyophilized from a mixture of 1,4-dioxane,0.1% aq. TFA and water.

[0133] Yield: 84 mg (TFA-salt)

[0134] MS-ESI: [M+H]⁺=531.2

[0135] HPLC: R_(t)=12.62 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 5

[0136] tert-Butyl5-amino-2-methylthio-4-(3-((N-(R-1-methoxycarbonyl-2-methyl-prop-1-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0137] D-Valine methyl ester hydrochloride (250 mg) was added to asolution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1 (h), 250 mg) and N,N-diisopropylethylamine (0.20 ml) in DCM(5 ml). After 16 h at room temperature, the mixture was diluted with DCM(50 ml), washed with sat. aq. NaHCO₃, dried (MgSO₄) and concentratedunder reduced pressure. The crude product was purified by HPLC using aLuna C-18 column with the following gradient: 0.1% aq. TFA+10% aq.ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compound was thenlyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0138] Yield: 77 mg (TFA-salt)

[0139] MS-ESI: [M+H]⁺=559.2

[0140] HPLC: R_(t)=13.22 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 6

[0141] tert-Butyl5-amino-2-methylthio-4-(3-((N,N-bis-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0142] N,N-bis-(2-methoxyethyl)-amine (400 mg) was added to a solutionof tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 250 mg) in DCM (5 ml). After 16 h at room temperature,the mixture was diluted with DCM (50 ml), washed with sat. aq. NaHCO₃,dried (MgSO₄) and concentrated under reduced pressure. The crude productwas purified by HPLC using a Luna C-18 column with the followinggradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min.The title compound was then lyophilized from a mixture of 1,4-dioxane,0.1% aq. TFA and water.

[0143] Yield: 166 mg (TFA-salt)

[0144] MS-ESI: [M+H]⁺=561.3

[0145] HPLC: R_(t)=13.62 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 7

[0146] tert-Butyl5-amino-2-methylthio-4-(3-((2,3-dihydroxy-prop-1-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0147] 3-Amino-2-hydroxy-propanol (250 mg) was added to a solution oftert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 250 mg) in DCM (5 ml). After 16 h at room temperature,the mixture was diluted with DCM (50 ml), washed with sat. aq. NaHCO₃,dried (MgSO₄) and concentrated under reduced pressure. The crude productwas purified by HPLC using a Luna C-18 column with the followinggradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min.The title compound was then lyophilized from a mixture of 1,4-dioxane,0.1% aq. TFA and water.

[0148] Yield: 164 mg (TFA-salt)

[0149] MS-ESI: [M+H]⁺=519.2

[0150] HPLC: R_(t)=12.62 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 8

[0151] tert-Butyl5-amino-2-methylthio-4-(3-((1,3-dihydroxyprop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0152] 2-amino-3-hydroxy propanol (250 mg) was added to a solution oftert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 250 mg) in DCM (5 ml). After 16 h at room temperature,the mixture was diluted with DCM (50 ml), washed with sat. aq. NaHCO₃,dried (MgSO₄) and concentrated under reduced pressure. The crude productwas purified by HPLC using a Luna C-18 column with the followinggradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min.The title compound was then lyophilized from a mixture of 1,4-dioxane,0.1% aq. TFA and water.

[0153] Yield: 117 mg (TFA-salt)

[0154] MS-ESI: [M+H]⁺=519.2

[0155] HPLC: R_(t)=12.62 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 9

[0156] tert-Butyl5-amino-2-phenyl-4-(3-((N-ethyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0157] (a). 5-Cyano-4-(3-nitrophenyl)-2-phenyl-6-hydroxy-pyrimidine

[0158] A mixture of benzamidine hydrochloride (16.4 g),3-nitrobenzaldehyde (15.1 g), ethyl cyanoacetate (11.2 ml) and potassiumcarbonate (16.6 g) in abs. EtOH (250 ml) was stirred at 60° C. for 8 h.The reaction mixture was cooled to 0° C. in an ice bath. The resultingprecipitate was filtered off, washed with abs. EtOH and heated in water(100° C.) until a clear solution was obtained. The solution was cooledto 50° C., acidified to pH 2 by adding 2N aq. HCl and cooled to 0° C. inan ice bath. The resulting precipitate was filtered off and washed withice water. Residual water was removed by coevaporation with 1,4-dioxane.

[0159] Yield: 15.0 g.

[0160] MS-ESI: [M+H]⁺=319.2

[0161] TLC: R_(f)=0.3, silica gel, DCM/MeOH=9/1 (v/v).

[0162] (b). 6-Chloro-5-cyano-4-(3-nitrophenyl)-2-phenyl-pyrimidine

[0163] POCl₃ (50 ml) was added to a stirred solution of5-cyano-4-(3-nitrophenyl)-2-phenyl-6-hydroxy-pyrimidine (example 9(a),15.0 g) and dimethylaniline (0.5 ml) in dry 1,4-dioxane p.a. (200 ml).After 3 h at 90° C., the warn mixture was filtered off and the filtratewas concentrated under reduced pressure. The residue was dissolved in1,4-dioxane and ice water was added. The resulting precipitate wasfiltered off and washed with water. Residual water was removed bycoevaporation with 1,4-dioxane.

[0164] Yield: 15.8 g

[0165] MS-ESI: [M+H]⁺=337.4

[0166] TLC: R_(f)=0.8, silica gel, heptane/EtOAc=3/2 (v/v).

[0167] (c). Ethyl5-cyano-4-(3-nitrophenyl)-2-phenyl-6-(ethoxycarbonylmethylthio)-pyrimidineDIPEA (8.71 ml) was added to a stirred solution of ethyl2-mercaptoacetate (5.15 ml) and6-chloro-5-cyano-4-(3-nitrophenyl)-2-phenyl-pyrimidine (example 9(b),15.8 g) in a mixture of EtOH (125 ml) and DCM (125 ml) under a nitrogenatmosphere. After 2 h at room temperature, the mixture was diluted withDCM until complete dissolution, washed with 0.5N aq. HCl, dried (MgSO₄)and concentrated under reduced pressure.

[0168] Yield: 19.7 g

[0169] MS-ESI: [M+H]⁺=421.2.

[0170] TLC: R_(f)=0.7, silica gel, heptane/EtOAc=3/2 (v/v).

[0171] (d). Ethyl5-amino-4-(3-nitrophenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxylate

[0172] DIPEA (20.0 ml) was added to a stirred solution of ethyl5-cyano-4-(3-nitrophenyl)-2-phenyl-6-(ethoxycarbonylmethylthio)-pyrimidine(example 9(c), 19.7 g) in a mixture of abs. EtOH (100 ml) and toluenep.a. (100 ml). After 48 h at 100° C., the mixture was cooled to 0° C.The resulting precipitate was filtered off, washed with cold EtOH anddried in vacuo at 40° C.

[0173] Yield: 17.0 g

[0174] MS-ESI: [M+H]⁺=421.2

[0175] TLC: R_(f)=0.5, silica gel, heptane/EtOAc=3/2 (v/v).

[0176] (e). Ethyl5-amino-4-(3-aminophenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxylate

[0177] A solution of tin (II) chloride (23.0 g) in abs. EtOH (250 ml)was added to a solution of ethyl5-amino-4-(3-nitrophenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxylate(example 9(d), 16.6 g) in 1,4-dioxane p.a (250 ml). 37% aq. HCl (6.9 ml)was added and the mixture was heated under reflux (90° C.) for 16 h. Themixture was allowed to cool to room temperature and concentrated underreduced pressure. The residue was suspended in EtOAc (500 ml). 4N aq.NaOH was added to obtain a pH of 10-11. The mixture was diluted byadding sat. aq. NaCl. The organic layer was separated, dried (MgSO₄) andconcentrated under reduced pressure.

[0178] Yield: 17.0 g

[0179] MS-ESI: [M+H]⁺=421.2

[0180] TLC: R_(f)=0.5, silica gel, heptane/EtOAc=3/2 (v/v).

[0181] (f).5-Amino-4-(3-aminophenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxylicacid

[0182] Potassium hydroxide (20.0 g) was added to a solution of ethyl5-amino-4-(3-aminophenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxylate(example 9(e), 17.0 g) in a mixture of 1,4-dioxane (210 ml) and water(80 ml). After 16 h at 90° C., the mixture was cooled to 0° C. Theresulting precipitate was filtered off, suspended in water (300 ml) andcooled to 0° C. The mixture was acidified to pH 3 by adding 2N aq.citric acid and stirred at 0° C. up to room temperature for 2 h. Theresulting precipitate was filtered off, washed with water and dried invacuo at 40° C.

[0183] Yield: 13.3 g

[0184] MS-ESI: [M+H]⁺=363.0

[0185] TLC: R_(f)=0.2, silica gel, DCM/MeOH=95/5 (v/v).

[0186] (g). tert-Butyl5-amino-4-(3-aminophenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxamide

[0187] DIPEA (15.3 ml), tert-butylamine (9.3 ml) and TBTU (14.1 g) wereadded to a mixture of5-amino-4-(3-aminophenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxylicacid (example 9(f), 13.3 g) in a mixture of DCM (250 ml) and DMF (50 ml)under a nitrogen atmosphere. After 3 h at room temperature, the mixturewas diluted with DCM and washed with sat. aq. NaHCO₃, 0.1N aq. HCl andsat. aq. NaCl. The organic layer was dried (MgSO₄) and concentratedunder reduced pressure. The crud product was purified by chromatographyon silica gel, using heptane/EtOAc=3/7 to 1/1 (v/v) as eluent.

[0188] Yield: 14.7 g

[0189] MS-ESI: [M+H]⁺=418.4

[0190] TLC: R_(f)=0.4, silica gel, heptane/EtOAc=3/2 (v/v).

[0191] (h). tert-Butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxamide

[0192] Bromoacetyl chloride (2.80 ml) was added dropwise to a solutionof tert-butyl5-amino-4-(3-aminophenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxamide(example 9(g), 5.8 g) and DIPEA (12.2 ml) in DCM (50 ml). After 3 h atroom temperature, the mixture was diluted with DCM, washed with sat. aq.NaHCO₃, dried (MgSO₄) and concentrated under reduced pressure. The crudeproduct was purified by chromatography on silica gel usingheptane/EtOAc=3/2 (v/v) as eluent. A 1:1 (mol/mol) mixture of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxamideand tert-butyl5-amino-4-(3-(2-chloroacetamido)-phenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxamidewas obtained.

[0193] Yield: 2.6 g

[0194] MS-ESI: [M+H]⁺=540.2, [M′+H]⁺=494.2

[0195] TLC: R_(f)=0.3, silica gel, heptane/EtOAc=3/2 (v/v).

[0196] (i). tert-Butyl5-amino-2-phenyl-4(3-((N-ethyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0197] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxamide(example 9(h), 500 mg) in DCM (5 ml) was added N-ethyl-2-amino-ethanol(500 mg). After stirring for 17 h at room temperature, the reactionmixture was diluted with DCM (100 ml), washed with aq. NaHCO₃ (1 M, 2×50ml), dried (MgSO₄) and concentrated in vacuo. The residue was purifiedby HPLC using a Luna C-18 column with the following gradient: 0.1% aq.TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compoundwas then lyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA andwater.

[0198] Yield: 271 mg (TFA-salt)

[0199] MS-ESI: [M+H]⁺=547.2

[0200] HPLC: R. =11.88 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 10

[0201] tert-Butyl5-amino-2-phenyl-4-(3-(N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0202] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-phenyl-thieno[2,3-d]pyrimidine-6-carboxamide(example 9(h), 500 mg) and N,N-diisopropylethyl amine (DiPEA, 1 ml) inDCM (5 ml) was added glycine methyl ester hydrochloride (700 mg). Afterstirring for 17 h at room temperature, the reaction mixture was dilutedwith DCM (100 ml), washed with aq. NaHCO₃ (1 M, 2×50 ml), dried (MgSO₄)and concentrated in vacuo. The residue was purified by HPLC using a LunaC-18 column with the following gradient: 0.1% aq. TFA+10% aq.ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compound was thenlyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0203] Yield: 321 mg (TFA-salt)

[0204] MS-ESI: [M+H]⁺=547.2

[0205] HPLC: R_(t)=12.54 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 11

[0206] tert-Butyl5-amino-2-(2-furyl)-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0207] (a) 2-amidinofuran

[0208] Cooled (0° C.) and saturated ethanolic HCl (40 ml) was added to acooled (ice-bath, 0° C.) reaction vessel, containing 2-furonitril (13ml). The resulting solution was allowed to reach ambient temperature andstirred under a nitrogen atmosphere for 48 h. After concentration of thereaction mixture in vacuo, the residue, containing the corresponding2-furyl ethyl imidate, was redissolved in ethanol (20 ml) and stirred at0° C. under a nitrogen atmosphere. Subsequently, saturated ethanolicammonia (40 ml) was added and the reaction mixture was stirred in asealed reaction vessel for 48 h. After filtration of the reactionmixture, the filtrate was concentrated under reduced pressure. The crudecompound was used without further purification in the next step.

[0209] Yield: 15.0 g

[0210] (b). 5-Cyano-4-(3-nitrophenyl)-2-(2-furyl)-6-hydroxy-pyrimidine

[0211] A mixture of 2-amidinofuran (example 11(a), 15 g),3-nitrobenzaldehyde (24 g), ethyl cyanoacetate (17 ml) and potassiumcarbonate (25 g) in abs. EtOH (300 ml) was stirred at 60° C. for 16 h.The reaction mixture was cooled to 0° C. in an ice bath. The resultingprecipitate was filtered off, washed with abs. EtOH and heated in water(100° C.) under stirring until a milky suspension was obtained. Thesuspension was cooled to 50° C., acidified to pH 2 by adding 2N aq. HCland cooled to 0° C. in an ice bath. The resulting precipitate wasfiltered off and washed with ice water. Residual water was removed bycoevaporation with 1,4-dioxane.

[0212] Yield: 16.0 g

[0213] MS-ESI: [M+H]⁺=309.2

[0214] TLC: R_(f)=0.3, silica gel, DCM/MeOH=9/1 (v/v).

[0215] (c). 6-Chloro-5-cyano-4-(3-nitrophenyl)-2-(2-furyl)-pyrimidine

[0216] POCl₃ (50 ml) was added to a stirred solution of5-cyano-4-(3-nitrophenyl)-2-(2-furyl)-6-hydroxy-pyrimidine (example 11(b), 16.0 g) and dimethylaniline (0.5 ml) in dry 1,4-dioxane p.a. (250ml). After 2 h at 90° C., the warm mixture was filtered off and thefiltrate was concentrated under reduced pressure. The residue wasdissolved in 1,4-dioxane and ice water was added. The resultingprecipitate was filtered off and washed with water. Residual water wasremoved by coevaporation with 1,4-dioxane.

[0217] Yield: 16.0 g

[0218] MS-ESI: [M+H]⁺=327.2

[0219] TLC: R_(f)=0.75, silica gel, heptane/EtOAc=3/2 (v/v).

[0220] (d). Ethyl5-Cyano-4-(3-nitrophenyl)-2-(2-furyl)-6-(ethoxycarbonylmethylthio)-pyrimidine

[0221] DIPEA (9.1 ml) was added to a stirred solution of ethyl2-mercaptoacetate (5.4 ml) and6-chloro-5-cyano-4-(3-nitrophenyl)-2-(2-furyl)-pyrimidine (example11(c), 16.0 g) in a mixture of EtOH (125 ml) and DCM (125 ml) under anitrogen atmosphere. After 2 h at room temperature, the mixture wasdiluted with DCM until complete dissolution, washed with 0.5N aq. HCl,dried (MgSO₄) and concentrated under reduced pressure.

[0222] Yield: 20.0 g

[0223] MS-ESI: [M+H]⁺=411.2.

[0224] TLC: R_(f)=0.7, silica gel, heptane/EtOAc=3/2 (v/v).

[0225] (e). Ethyl5-amino-4-(3-nitrophenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxylate

[0226] DIPEA (20.0 ml) was added to a stirred solution of ethyl5-cyano-4-(3-nitrophenyl)-2-(2-furyl)-6-(ethoxycarbonylmethylthio)-pyrimidine(example 11(d), 20 g) in a mixture of abs. EtOH (100 ml) and toluenep.a. (100 ml). After 48 h at 100° C., the mixture was cooled to 0° C.The resulting precipitate was filtered off, washed with cold EtOH anddried in vacuo at 40° C.

[0227] Yield: 20 g

[0228] MS-ESI: [M+H]⁺=411.2

[0229] TLC: R_(f)=0.6, silica gel, heptane/EtOAc=3/2 (v/v).

[0230] (f). Ethyl5-amino-4-(3-aminophenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxylate

[0231] A solution of tin (II) chloride (28.0 g) in abs. EtOH (250 ml)was added to a solution of ethyl5-amino-4-(3-nitrophenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxylate(example 11(e), 20 g) in 1,4-dioxane p.a. (250 ml). 37% aq. HCl (8.5 ml)was added and the mixture was heated under reflux (90° C.) for 16 h. Themixture was allowed to cool to room temperature and concentrated underreduced pressure. The residue was suspended in EtOAc (500 ml). 4N aq.NaOH was added to obtain a pH of 10-11. The mixture was diluted byadding sat. aq. NaCl. The organic layer was separated, dried (MgSO₄) andconcentrated under reduced pressure.

[0232] Yield: 17.5 g

[0233] MS-ESI: [M+H]⁺=381.2

[0234] TLC: R_(f)=0.4, silica gel, heptane/EtOAc=3/2 (v/v).

[0235] (g).5-Amino-4-(3-aminophenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxylicacid

[0236] Potassium hydroxide (23.0 g) was added to a solution of ethyl5-amino-4-(3-aminophenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxylate(example 11(f), 17.5 g) in a mixture of 1,4-dioxane (210 ml) and water(80 ml). After 8 h at 90° C., the mixture was cooled to 0° C. Theresulting precipitate was filtered off, suspended in water (300 ml) andcooled to 0° C. The mixture was acidified to pH 3 by adding 2N aq.citric acid and stirred at 0° C. up to room temperature for 2 h. Theresulting precipitate was filtered off, washed with water and dried invacuo at 40° C.

[0237] Yield: 16.9 g

[0238] MS-ESI: [M+H]⁺=353.2

[0239] TLC: R_(f)=0.2, silica gel, DCM/MeOH=95/5 (v/v).

[0240] (h). tert-Butyl5-amino-4-(3-aminophenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0241] DIPEA (19.2 ml), tert-butylamine (11.6 ml) and TBTU (17.7 g) wereadded to a solution of5-amino-4-(3-aminophenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxylicacid (example 11(g), 16.9 g) in a mixture of DCM (250 ml) and DMF (50ml) under a nitrogen atmosphere. After 3 h at room temperature, asubstantial amount of yellow precipitate had been formed, which wasfiltered off. The residue was washed with diethyl ether and dried invacuo at 40° C.

[0242] Yield: 18.0 g

[0243] MS-ESI: [M+H]⁺=408.2

[0244] TLC: R_(f)=0.4, silica gel, heptane/EtOAc=3/2 (v/v).

[0245] (i). tert-Butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0246] Bromoacetyl chloride (100 l) was added dropwise to a solution oftert-butyl5-amino-4-(3-aminophenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 11 (h), 250 mg) and DIPEA (0.5 ml) in DCM (5 ml). After 3 h atroom temperature, the mixture was diluted with DCM, washed with sat. aq.NaHCO₃, dried (MgSO₄) and concentrated under reduced pressure. The crudeproduct was purified by chromatography on silica gel usingheptane/EtOAc=3/2 (v/v) as eluent. A 1:1 (mol/mol) mixture of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamideand tert-butyl5-amino-4-(3-(2-chloroacetamido)-phenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamidewas obtained.

[0247] Yield: 124 mg

[0248] MS-ESI: [M+H]⁺=540.2, [M′+H]⁺=494.2

[0249] TLC: R_(f)=0.3, silica gel, heptane/EtOAc=3/2 (v/v).

[0250] (i). tert-Butyl5-amino-2-(2-furyl)-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0251] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 11(i), 130 mg) in DCM (5 ml) was added N-methyl-2-amino-ethanol(200 mg). After stirring for 17 h at room temperature, the reactionmixture was diluted with DCM (50 ml), washed with aq. NaHCO₃ (1 M, 2×10ml), dried (MgSO₄) and concentrated in vacuo. The residue was purifiedby HPLC using a Luna C-18 column with the following gradient: 0.1% aq.TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compoundwas then lyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA andwater.

[0252] Yield: 81 mg (TFA-salt)

[0253] MS-ESI: [M+H]⁺=523.2

[0254] HPLC: R_(t)=11.01 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection V=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 12

[0255] tert-Butyl5-amino-2-(2-furyl)-4-(3-(N-(1-hydroxy-2-methyl-prop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0256] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 11(i), 130 mg) in DCM (5 ml) was added2-amino-2-methyl-propanol (260 mg). After stirring for 17 h at roomtemperature, the reaction mixture was diluted with DCM (50 ml), washedwith aq. NaHCO₃ (1 M, 2×10 ml), dried (MgSO₄) and concentrated in vacuo.The residue was purified by HPLC using a Luna C-18 column with thefollowing gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in30 min. The title compound was then lyophilized from a mixture of1,4-dioxane, 0.1% aq. TFA and water.

[0257] Yield: 54 mg (TFA-salt)

[0258] MS-ESI: [M+H]⁺=537.2

[0259] HPLC: R_(t)=11.15 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 13

[0260] tert-Butyl5-amino-2-(2-furyl)-4-(3-(N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0261] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-furyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 11(i), 130 mg) and DIPEA (0.5 ml) in DCM (5 ml) was addedglycine methyl ester hydrochloride (324 mg). After stirring for 17 h atroom temperature, the reaction mixture was diluted with DCM (50 ml),washed with aq. NaHCO₃ (1 M, 2×10 ml), dried (MgSO₄) and concentrated invacuo. The residue was purified by HPLC using a Luna C-18 column withthe following gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90(v/v) in 30 min. The title compound was then lyophilized from a mixtureof 1,4-dioxane, 0.1% aq. TFA and water.

[0262] Yield: 74 mg (TFA-salt)

[0263] MS-ESI: [M+H]⁺=537.2

[0264] HPLC: R_(t)=12.09 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 m/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 14

[0265] tert-Butyl5-amino-2-(2-thienyl)-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0266] (a). 5-Cyano-4-(3-nitrophenyl)-2-(2-thienyl)-6-hydroxy-pyrimidine

[0267] A mixture of 2-amidinothiophene hydrochloride (10.0 g),3-nitrobenzaldehyde (9.7 g), ethyl cyanoacetate (6.81 ml) and potassiumcarbonate (10.1 g) in abs. EtOH (200 ml) was stirred at 60° C. for 8 h.The reaction mixture was cooled to 0° C. in an ice bath, filtered,washed with abs. EtOH and the residue was dissolved in water (100° C.).The solution was cooled to 50° C., acidified with 2N aq. HCl to pH 2 andcooled to 0° C. in an ice bath. The resulting precipitate was filteredoff and washed with ice water. Residual water was removed bycoevaporation with 1,4-dioxane.

[0268] Yield: 10.0 g

[0269] MS-ESI: [M+H]⁺=325.0

[0270] TLC: R_(f)=0.3, silica gel, DCM/MeOH=9/1 (v/v).

[0271] (b). 6-Chloro-5-cyano-4-(3-nitrophenyl)-2-(2-thienyl)-pyrimidine

[0272] POCl₃ (30 ml) was added to a stirred solution of5-cyano-4-(3-nitrophenyl)-2-(2-thienyl)-6-hydroxy-pyrimidine (example14(a), 10.0 g) and dimethylaniline (a few drops) in dry 1,4-dioxane (150ml). After 3 h at 90° C., the mixture was cooled to room temperature andconcentrated under reduced pressure. The residue was dissolved in1,4-dioxane and ice water was cautiously added. The resultingprecipitate was filtered off and washed with water. Residual water wasremoved by coevaporation with 1,4-dioxane and drying in vacuo at 40° C.

[0273] Yield: 9.8 g

[0274] MS-ESI: [M+H]⁺=343.4

[0275] TLC: =0.8, silica gel, heptane/EtOAc=3/2 (v/v).

[0276] (c). Ethyl5-cyano-4-(3-nitrophenyl)-2-(2-thienyl)-6-(ethoxycarbonylmethylthio)-pyrimidine

[0277] DIPEA (5.57 ml) was added to a stirred solution of ethyl2-mercaptoacetate (3.28 ml) and6-chloro-5-cyano-4-(3-nitrophenyl)-2-(2-thienyl)-pyrimidine (example14(b), 9.8 g) in a mixture of EtOH (80 ml) and DCM (80 ml) under anitrogen atmosphere. After 2 h at room temperature, the mixture wasdiluted with DCM until complete dissolution, washed with 0.5N aq. HCl,dried (MgSO₄) and concentrated under reduced pressure.

[0278] Yield: 12.9 g

[0279] MS-ESI: [M+H]⁺=427.2

[0280] TLC: R_(f)=0.7, silica gel, heptane/EtOAc=3/2 (v/v).

[0281] (d). Ethyl5-amino-4-(3-nitrophenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxylate

[0282] DIPEA (13.0 ml) was added to a stirred solution of ethyl5-cyano-4-(3-nitrophenyl)-2-(2-thienyl)-6-(ethoxycarbonylmethylthio)-pyrimidine(example 14(c), 12.9 g) in a mixture of abs. EtOH (75 ml) and toluenep.a. (75 ml). After 48 h at 100° C., the mixture was cooled to 0° C. Theresulting precipitate was filtered off, washed with cold EtOH and driedin vacuo at 40° C.

[0283] Yield: 11.0 g

[0284] MS-ESI: [M+H]⁺=427.2

[0285] TLC: R_(f)=0.6, silica gel, heptane/EtOAc=3/2 (v/v).

[0286] (e). Ethyl5-amino-4-(3-aminophenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxylate

[0287] A solution of tin (II) chloride (15 g) in abs. EtOH (150 ml) wasadded to a solution of ethyl5-amino-4-(3-nitrophenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxylate(example 14(d), 10.86 g) in 1,4-dioxane (150 ml). 37% aq. HCl (4.5 ml)was added and the mixture was heated under reflux for 16 h. The mixturewas allowed to cool to room temperature and concentrated under reducedpressure. The residue was suspended in EtOAc (400 ml) and THF was addeduntil complete dissolution. 4N aq. NaOH was added to obtain a pH of10-11. The mixture was diluted by adding sat. aq. NaCl. The organiclayer was separated, dried (MgSO₄) and concentrated under reducedpressure.

[0288] Yield: 12.0 g

[0289] MS-ESI: [M+H]⁺=397.2

[0290] TLC: R_(f)=0.4, silica gel, heptane/EtOAc=3/2 (v/v).

[0291] (f).5-Amino-4-(3-aminophenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxylicacid

[0292] Potassium hydroxide (13 g) was added to a solution of ethyl5-amino-4-(3-aminophenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxylate(example 14(e), 10.1 g) in a mixture of 1,4-dioxane (150 ml) and water(50 ml). After 16 h at 90° C., the mixture was cooled to 0° C. Theresulting precipitate was filtered off, suspended in water (180 ml) andcooled to 0° C. The mixture was acidified to pH 3 by adding 2N aq.citric acid and stirred at 0° C. for 2 h. The resulting precipitate wasfiltered off, washed with water and dried in vacuo at 40° C.

[0293] Yield: 6.3 g

[0294] MS-ESI: [M+H]⁺=369.2

[0295] TLC: R=0.2, silica gel, DCM/MeOH=95/5 (v/v).

[0296] (g). tert-Butyl5-amino-4-(3-aminophenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0297] DIPEA (7.1 ml), tert-butylamine (4.3 ml) and TBTU (6.6 g) wereadded to a mixture of5-amino-4-(3-aminophenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxylicacid (example 14(f), 6.3 g) in a mixture of DCM (125 ml) and DMF(N,N-dimethylformamide) (25 ml) under a nitrogen atmosphere. After 3 hat room temperature, the mixture was diluted with DCM and washed withsat. aq. NaHCO₃, 0.1 N aq. HCl and sat. aq. NaCl. The organic layer wasdried (MgSO₄) and concentrated under reduced pressure. The crude productwas purified by chromatography on silica gel, using heptane/EtOAc=3/7 to1/1 (v/v) as eluent.

[0298] Yield: 6.45 g

[0299] MS-ESI: [M+H]⁺=424.2

[0300] TLC: R_(f)=0.3, silica gel, heptane/EtOAc=3/2 (v/v).

[0301] (h). tert-Butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0302] Bromoacetyl chloride (2.40 ml) was added to a solution oftert-butyl5-amino-4-(3-aminophenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 14(g), 5.0 g) and DIPEA (10.5 ml) in DCM (50 ml). After 3 h atroom temperature, the mixture was diluted with DCM, washed with sat. aq.NaHCO₃, dried (MgSO₄) and concentrated under reduced pressure. The crudeproduct was purified by chromatography on silica gel usingheptane/EtOAc=3/2 (v/v) as eluent. A mixture of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamideand tert-butyl5-amino-4-(3-(2-chloroacetamido)-phenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamidewas obtained.

[0303] Yield: 3.0 g

[0304] MS-ESI: [M+H]⁺=546.2, [M′+H]⁺=500.2

[0305] TLC: R_(f)=0.2, silica gel, toluene/EtOAc=7/1 (v/v).

[0306] (i). tert-Butyl5-amino-2-(2-thienyl)-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0307] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 14(h), 100 mg) in DCM (5 ml) was added N-methyl-2-aminoethanol(140 mg). After stirring for 17 h at room temperature, the reactionmixture was diluted with DCM (50 ml), washed with aq. NaHCO₃ (1 M, 2×10ml), dried (MgSO₄) and concentrated in vacuo. The residue was purifiedby HPLC using a Luna C-18 column with the following gradient: 0.1% aq.TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compoundwas then lyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA andwater.

[0308] Yield: 59 mg (TFA-salt)

[0309] MS-ESI: [M+H]⁺=539.2

[0310] HPLC: R_(t)=11.01 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 15

[0311] tert-Butyl5-amino-2-(2-thienyl)-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0312] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 14(h), 100 mg) and DIPEA (0.5 ml) in DCM (5 ml) was addedglycine methyl ester hydrochloride (250 mg). After stirring for 17 h atroom temperature, the reaction mixture was diluted with DCM (50 ml),washed with aq. NaHCO₃ (1 M, 2×10 ml), dried (MgSO₄) and concentrated invacuo. The residue was purified by HPLC using a Luna C-18 column withthe following gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90(v/v) in 30 min. The title compound was then lyophilized from a mixtureof 1,4-dioxane, 0.1% aq. TFA and water.

[0313] Yield: 74 mg (TFA-salt)

[0314] MS-ESI: [M+H]⁺=553.0

[0315] HPLC: R_(t)=12.57 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 16

[0316] tert-Butyl5-amino-2-(2-thienyl)-4-(3-((N,N-di-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0317] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(2-thienyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 14(h), 100 mg) in DCM (5 ml) was addedN,N-di-(2-methoxyethyl)-amine (200 mg). After stirring for 17 h at roomtemperature, the reaction mixture was diluted with DCM (50 ml), washedwith aq. NaHCO₃ (1 M, 2×10 ml), dried (MgSO₄) and concentrated in vacuo.The residue was purified by HPLC using a Luna C-18 column with thefollowing gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in30 min. The title compound was then lyophilized from a mixture of1,4-dioxane, 0.1% aq. TFA and water.

[0318] Yield: 59 mg (TFA-salt)

[0319] MS-ESI: [+H]⁺=597.4

[0320] HPLC: R_(t)=13.84 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 17

[0321] tert-Butyl5-amino-2-ethylamino-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0322] (a). tert-Butyl5-amino-2-methanesulfinyl-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0323] To a stirred solution of tert-butyl5-amino-2-methylthio-4-(3-((N-ethyl-N-(2methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 4, 1.0 g) in trifluoroacetic acid (TFA, 25 ml) was added3-chloroperbenzoic acid (m-CPBA, 1.0 g). After 17 h, the reactionmixture was concentrated under reduced pressure at ambient temperature(20° C.), redissolved in DCM (100 ml), carefully washed with sat. aq.NaHCO₃ (2×50 ml) and water (50 ml), dried (MgSO₄) and concentrated invacuo. The crude residue was used without further purification in thenext step.

[0324] Yield: 820 mg

[0325] MS-ESI: [M+H]⁺=547.3

[0326] TLC: R_(f)=0.2, silica gel, DCM/MeOH=9/1 (v/v).

[0327] (b). tert-Butyl5-amino-2-ethylamino-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0328] Ethyl amine hydrochloride (150 mg) was added to a stirredsolution of tert-butyl5-amino-2-methanesulfinyl-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 17(a), 100 mg) and DiPEA (0.5 ml) in 1,4-dioxane (5 ml) and thereaction mixture was heated to 60° C. for 3 h. After concentration ofthe reaction mixture under reduced pressure, the residue was taken up inDCM (50 ml) and washed with brine (1 M, 25 ml) and water (25 ml).Subsequently, the organic layer was dried (MgSO₄) and concentrated invacuo. The thus obtained residue was purified by HPLC using a Luna C-18column with the following gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10to 10/90 (v/v) in 30 min. The title compound was then lyophilized from amixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0329] Yield: 36 mg (TFA-salt)

[0330] MS-ESI: [M+H]⁺=528.4

[0331] HPLC: R_(t)=10.21 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 18

[0332] tert-Butyl5-amino-2-(N,N-dimethylamino)-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0333] (a). tert-Butyl5-amino-2-methanesulfinyl-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0334] To a stirred solution of tert-butyl5-amino-2-methylthio-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(i), 1.0 g) in trifluoroacetic acid (TFA, 25 ml) was added3-chloroperbenzoic acid (m-CPBA, 1.0 g). After 17 h, the reactionmixture was concentrated under reduced pressure at ambient temperature(20° C.), redissolved in DCM (100 ml), carefully washed with sat. aq.NaHCO₃ (2×50 ml) and water (50 ml), dried (MgSO₄) and concentrated invacuo. The crude residue was used without further purification in thenext step.

[0335] Yield: 910 mg

[0336] MS-ESI: [M+H]⁺=519.6

[0337] TLC: R_(f)=0.15, silica gel, DCM/MeOH=9/1 (v/v).

[0338] (b). tert-Butyl5-amino-2-(N,N-dimethylamino)-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0339] Dimethyl amine hydrochloride (150 mg) was added to a stirredsolution of tert-butyl5-amino-2-methanesulfinyl-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 18(a), 100 mg) and DIPEA (0.5 ml) in 1,4-dioxane (5 ml) and thereaction mixture was heated to 60° C. for 3 h. After concentration ofthe reaction mixture under reduced pressure, the residue was taken up inDCM (50 ml) and washed with brine (1 M, 25 ml) and water (25 ml).Subsequently, the organic layer was dried (MgSO₄) and concentrated invacuo. The thus obtained residue was purified by HPLC using a Luna C-18column with the following gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10to 10/90 (v/v) in 30 min. The title compound was then lyophilized from amixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0340] Yield: 36 mg (TFA-salt)

[0341] MS-ESI: [M+H]⁺=500.2

[0342] HPLC: R_(t)=10.03 min, column Luna C-18(2), 3 lm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 19

[0343] tert-Butyl5-amino-2-ethylamino-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0344] (a). tert-Butyl5-amino-2-methanesulfinyl-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0345] To a stirred solution of tert-butyl5-amino-2-methylthio-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 3, 1.0 g) in trifluoroacetic acid (TFA, 25 ml) was added3-chloroperbenzoic acid (m-CPBA, 1.0 g). After 17 h, the reactionmixture was concentrated under reduced pressure at ambient temperature(20° C.), redissolved in DCM (100 ml), carefully washed with sat. aq.NaHCO₃ (2×50 ml) and water (50 ml), dried (MgSO₄) and concentrated invacuo. The crude residue was used without further purification in thenext step.

[0346] Yield: 680 mg

[0347] MS-ESI: [M+H]⁺=533.6

[0348] TLC: R_(f)=0.17, silica gel, DCM/MeOH=9/1 (v/v).

[0349] (b). tert-Butyl5-amino-2-ethylamino-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0350] Ethyl amine hydrochloride (150 mg) was added to a stirredsolution of tert-butyl5-amino-2-methanesulfinyl-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 19(a), 100 mg) and DiPEA (0.5 ml) in 1,4-dioxane (5 ml) and thereaction mixture was heated to 60° C. for 3 h. After concentration ofthe reaction mixture under reduced pressure, the residue was taken up inDCM (50 ml) and washed with brine (1 M, 25 ml) and water (25 ml).Subsequently, the organic layer was dried (MgSO₄) and concentrated invacuo. The thus obtained residue was purified by HPLC using a Luna C-18column with the following gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10to 10/90 (v/v) in 30 min. The title compound was then lyophilized from amixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0351] Yield: 57 mg (TFA-salt)

[0352] MS-ESI: [M+H]⁺=514.2

[0353] HPLC: R_(t)=12.56 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 20

[0354] tert-Butyl5-amino-2-isopropylamino-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0355] Isopropyl amine (150 mg) was added to a stirred solution oftert-butyl5-amino-2-methanesulfinyl-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 17(a), 100 mg) and DiPEA (0.5 ml) in 1,4-dioxane (5 ml) and thereaction mixture was heated to 60° C. for 3 h. After concentration ofthe reaction mixture under reduced pressure, the residue was taken up inDCM (50 ml) and washed with brine (1 M, 25 ml) and water (25 ml).Subsequently, the organic layer was dried (MgSO₄) and concentrated invacuo. The thus obtained residue was purified by HPLC using a Luna C-18column with the following gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10to 10/90 (v/v) in 30 min. The title compound was then lyophilized from amixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0356] Yield: 57 mg (TFA-salt)

[0357] MS-ESI: [M+H]⁺=542.4

[0358] HPLC: R_(t)=11.01 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 21

[0359] tert-Butyl5-amino-2-allylamino-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0360] Allyl amine (200 mg) was added to a stirred solution oftert-butyl5-amino-2-methanesulfinyl-4-(3-((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 19(a), 100 mg) and DIPEA (0.5 ml) in 1,4-dioxane (5 ml) and thereaction mixture was heated to 60° C. for 3 h. After concentration ofthe reaction mixture under reduced pressure, the residue was taken up inDCM (50 ml) and washed with brine (1 M, 25 ml) and water (25 ml).Subsequently, the organic layer was dried (MgSO₄) and concentrated invacuo. The thus obtained is residue was purified by HPLC using a LunaC-18 column with the following gradient: 0.1% aq. TFA+10% aq.ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compound was thenlyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0361] Yield: 65 mg (TFA-salt)

[0362] MS-ESI: [M+H]⁺=526.4

[0363] HPLC: R. =13.18 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 22

[0364] tert-Butyl5-amino-2-methoxy-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0365] Potassium tert-butoxide (100 mg) was added to a stirred solutionof tert-butyl5-amino-2-methanesulfinyl-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 17(a), 200 mg) in methanol (5 ml) and the reaction mixture washeated to 50° C. for 3 h. After concentration of the reaction mixtureunder reduced pressure, the residue was taken up in DCM (50 ml) andwashed with aq. ammonium chloride (1 M, 25 ml), brine (1 M, 25 ml) andwater (25 ml). Subsequently, the organic layer was dried (MgSO₄) andconcentrated in vacuo. The thus obtained residue was purified by HPLCusing a Luna C-18 column with the following gradient: 0.1% aq. TFA+10%aq. ACN/ACN 90/10 to 10/90 (v/v) in 30 min. The title compound was thenlyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0366] Yield: 92 mg (TFA-salt)

[0367] MS-ESI: [M+H]⁺=515.4

[0368] HPLC: R_(t)=12.21 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 23

[0369] tert-Butyl5-amino-2-allyloxy-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0370] Potassium tert-butoxide (100 mg) was added to a stirred solutionof tert-butyl5-amino-2-methanesulfinyl-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 17(a), 200 mg) in allyl alcohol (5 ml) and the reaction mixturewas heated to 50° C. for 3 h. After concentration of the reactionmixture under reduced pressure, the residue was taken up in DCM (50 ml)and washed with aq. ammonium chloride (1 M, 25 ml), brine (1 M, 25 ml)and water (25 ml). Subsequently, the organic layer was dried (MgSO₄) andconcentrated in vacuo. The thus obtained residue was purified by HPLCusing a Luna C-18 column with the following gradient: 0.1% aq. TFA+10%aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compound was thenlyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0371] Yield: 63 mg (TFA-salt)

[0372] MS-ESI: [M+H]⁺=541.4

[0373] HPLC: R_(t) 12.71 min, column Luna C-18(2), 3 μm, 100×2:0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 24

[0374] tert-Butyl5-amino-2-isopropoxy-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0375] Potassium tert-butoxide (100 mg) was added to a stirred solutionof tert-butyl5-amino-2-methanesulfinyl-4-(3-((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 17(a), 200 mg) in isopropanol (5 ml) and the reaction mixturewas heated to 50° C. for 3 h. After concentration of the reactionmixture under reduced pressure, the residue was taken up in DCM (50 ml)and washed with aq. ammonium chloride (1 M, 25 ml), brine (1 M, 25 ml)and water (25 ml). Subsequently, the organic layer was dried (MgSO₄) andconcentrated in vacuo. The thus obtained residue was purified by HPLCusing a Luna C-18 column with the following gradient: 0.1% aq. TFA+10%aq. ACN/ACN=90/10 to 10/90 (v/v) in 30 min. The title compound was thenlyophilized from a mixture of 1,4-dioxane, 0.1% aq. TFA and water.

[0376] Yield: 32 mg (TFA-salt)

[0377] MS-ESI: [M+H]⁺=543.4

[0378] HPLC: R_(t)=12.93 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 25

[0379] tert-Butyl5-amino-2-(4-pyridyl)-4-(3-((N-(1-hydroxy-2-methyl-prop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0380] (a). 5-Cyano-4-(3-nitrophenyl)-2-(4-pyridyl)-6-hydroxy-pyrimidine

[0381] A mixture of 4-amidino-pyridine hydrochloride (16.5 g),3-nitrobenzaldehyde (15.1 g), ethyl cyanoacetate (11.2 ml) and potassiumcarbonate (16.6 g) in abs. EtOH (250 ml) was stirred at 60° C. for 16 h.The reaction mixture was cooled to 0° C. in an ice bath. The resultingprecipitate was filtered off, washed with abs. EtOH and heated in water(100° C.) until a clear solution was obtained. The solution was cooledto 50° C., acidified to pH 2 by adding 2N aq. HCl and cooled to 0° C. inan ice bath. The resulting precipitate was filtered off and washed withice water. Residual water was removed by coevaporation with 1,4-dioxane.

[0382] Yield: 18.3 g

[0383] MS-ESI: [M+H]⁺=320.2

[0384] TLC: R_(f)=0.2, silica gel, DCM/MeOH=9/1 (v/v)

[0385] (b). 6-Chloro-5-cyano-4-(3-nitrophenyl)-2-(4-pyridyl)-pyrimidine

[0386] POCl₃ (50 ml) was added to a stirred solution of5-cyano-4-(3-nitrophenyl)-2-(4-pyridyl)-6-hydroxy-pyrimidine (example25(a), 18.3 g) and dimethylaniline (0.5 ml) in dry 1,4-dioxane p.a. (200ml). After 3 h at 90° C., the warm mixture was filtered off and thefiltrate was concentrated under reduced pressure. The residue wasdissolved in 1,4-dioxane and ice water was added. The resultingprecipitate was filtered off and washed with water. Residual water wasremoved by coevaporation with 1,4-dioxane.

[0387] Yield: 17.2 g

[0388] MS-ESI: [M+H]⁺=338.4

[0389] TLC: R_(f)=0.7, silica gel, heptane/EtOAc=3/2 (v/v)

[0390] (c). Ethyl5-cyano-4-(3-nitrophenyl)-2-(4-pyridyl)-6-(ethoxycarbonylmethylthio)-pyrimidine

[0391] DIPEA (9.8 ml) was added to a stirred solution of ethyl2-mercaptoacetate (5.7 ml) and6-chloro-5-cyano-4-(3-nitrophenyl)-2-(4-pyridyl)-pyrimidine (example25(b), 17.2 g) in a mixture of EtOH (125 ml) and DCM (125 ml) under anitrogen atmosphere. After 2 h at room temperature, the mixture wasdiluted with DCM until complete dissolution, washed with 0.5N aq. HCl,dried (MgSO₄) and concentrated under reduced pressure.

[0392] Yield: 20.5 g

[0393] MS-ESI: [M+H]⁺=422.0

[0394] TLC: R_(f)=0.6, silica gel, heptane/EtOAc=3/2 (v/v)

[0395] (d). Ethyl5-amino-4-(3-nitrophenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxylate

[0396] DIPEA (20.0 ml) was added to a stirred solution of ethyl5-cyano-4-(3-nitrophenyl)-2-(4-pyridyl)-6-(ethoxycarbonylmethylthio)-pyrimidine(example 25(c), 20.5 g) in a mixture of abs. EtOH (100 ml) and toluenep.a. (100 ml). After 48 h at 100° C., the mixture was cooled to 0° C.The resulting precipitate was filtered off, washed with cold EtOH anddried in vacuo at 40° C.

[0397] Yield: 15.7 g

[0398] MS-ESI: [M+H]⁺=422.2

[0399] TLC: R_(f)=0.5, silica gel, heptane/EtOAc=3/2 (v/v).

[0400] (e). Ethyl5-amino-4-(3-aminophenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxylate

[0401] A solution of tin (II) chloride (21.0 g) in abs. EtOH (250 ml)was added to a solution of ethyl5-amino-4-(3-nitrophenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxylate(example 25(d), 15.7 g) in 1,4-dioxane p.a. (250 ml). 37% aq. HCl (6.9ml) was added and the mixture was heated under reflux (90° C.) for 16 h.The mixture was allowed to cool to room temperature and concentratedunder reduced pressure. The residue was suspended in EtOAc (500 ml). 4Naq. NaOH was added to obtain a pH of 10-11. The mixture was diluted byadding sat. aq. NaCl. The organic layer was separated, dried (MgSO₄) andconcentrated under reduced pressure.

[0402] Yield: 12.0 g

[0403] MS-ESI: [M+H]⁺=392.2

[0404] TLC: R_(f)=0.3, silica gel, heptane/EtOAc=3/2 (v/v).

[0405] (f).5-Amino-4-(3-aminophenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxylicacid

[0406] Potassium hydroxide (15.7 g) was added to a solution of ethyl5-amino-4-(3-aminophenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxylate(example 25(e), 12.0 g) in a mixture of 1,4-dioxane (210 ml) and water(80 ml). After 16 h at 90° C., the mixture was cooled to 0° C. Theresulting precipitate was filtered off, suspended in water (300 ml) andcooled to 0° C. The mixture was acidified to pH 3 by adding 2N aq.citric acid and stirred at 0° C. up to room temperature for 2 h. Theresulting precipitate was filtered off, washed with water and dried invacuo at 40° C.

[0407] Yield: 12.0 g

[0408] MS-ESI: [M+H]⁺=364.2

[0409] TLC: R=0.1, silica gel, DCM/MeOH=95/5 (v/v).

[0410] (g). tert-Butyl5-amino-4-(3-aminophenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0411] DIPEA (12.9 ml), tert-butylamine (7.8 ml) and TBTU (11.9 g) wereadded to a mixture of5-amino-4-(3-aminophenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxylicacid (example 25(f), 12.0 g) in a mixture of DCM (250 ml) and DMF (50ml) under a nitrogen atmosphere. After 2 h at room temperature, themixture was diluted with DCM and washed with sat. aq. NaHCO₃, O. 1N aq.HCl and sat. aq. NaCl. The organic layer was dried (MgSO₄) andconcentrated under reduced pressure. The crude product was purified bychromatography on silica gel, using dichloromethane/methanol=1/0 to 95/5(v/v) as eluent.

[0412] Yield: 12.2 g

[0413] MS-ESI: [M+H]⁺=419.4

[0414] TLC: R_(t)=0.3, silica gel, heptane/EtOAc=3/2 (v/v).

[0415] (h). tert-Butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0416] A solution of bromoacetyl bromide (0.59 ml) in THF (25 ml) wasadded dropwise to a solution of tert-butyl5-amino-4-(3-aminophenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 25(g), 2.0 g) and N,N-dimethyl aniline (3.0 ml) in THF (50 ml).After 30 min at room temperature, the mixture was concentrated underreduced pressure, subsequently dissolved in DCM (100 ml), washed withsat. aq. NaHCO₃, dried (MgSO₄) and concentrated in vacuo. The residuewas used without further purification in the next step.

[0417] Yield: 2.3 g

[0418] MS-ESI: [M+H]⁺=539.2

[0419] TLC: R_(f)=0.3, silica gel, heptane/EtOAc=3/2 (v/v).

[0420] (i). tert-Butyl5-amino-2-(4-pyridyl)-4-(3-((N-(1-hydroxy-2-methyl-prop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0421] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 25(h), 200 mg) in DCM (5 ml) was added2-amino-2-methyl-propanol (300 mg). After stirring for 17 h at roomtemperature, the reaction mixture was diluted with DCM (100 ml), washedwith aq. NaHCO₃ (1 M, 2×50 ml), dried (MgSO₄) and concentrated in vacuo.The residue was purified by HPLC using a Luna C-18 column with thefollowing gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in30 min. The title compound was then lyophilized from a mixture of1,4-dioxane, 0.1% aq. TFA and water.

[0422] Yield: 73 mg (TFA-salt)

[0423] MS-ESI: [M+H]⁺=548.2

[0424] HPLC:R_(t)=9.65 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 26

[0425] tert-Butyl5-amino-2-(4-pyridyl)₄-(3-((N,N-bis-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

[0426] To a stirred solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-(4-pyridyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 25h), 200 mg) in DCM (5 ml) was addedbis-(2-methoxyethyl)-amine (300 mg). After stirring for 17 h at roomtemperature, the reaction mixture was diluted with DCM (100 ml), washedwith aq. NaHCO₃ (1 M, 2×50 ml), dried (MgSO₄) and concentrated in vacuo.The residue was purified by HPLC using a Luna C-18 column with thefollowing gradient: 0.1% aq. TFA+10% aq. ACN/ACN=90/10 to 10/90 (v/v) in30 win. The title compound was then lyophilized from a mixture of1,4-dioxane, 0.1% aq. TFA and water.

[0427] Yield: 170 mg (TFA-salt)

[0428] MS-ESI: [M+H]⁺=592.2

[0429] HPLC: R_(t)=12.02 min, column Luna C-18(2), 3 μm, 100×2.0 mm,detection UV=210 nm, oven temperature=40° C., flow=0.25 ml/min, eluentphosphate buffer 50 mM pH 2.1/water/ACN=10/80/10 to 10/10/80 (v/v/v),run time=20 min.

Example 27

[0430] CHO-LH and CHO-FSH In Vitro Bioactivity

[0431] LH agonistic activity of compounds were tested in Chinese HamsterOvary (CHO) cells stably transfected with the human LH receptor andcotransfected with a cAMP responsive element (CRE)/promotor directingthe expression of a firefly luciferase reporter gene. Binding of ligandto the Gs-coupled LH receptor will result in an increase of cAMP, whichin turn will induce an increased transactivation of the luciferasereporter construct. The luciferase signal was quantified using aluminescence counter. For test compounds, EC₅₀ values (concentration oftest compound causing half-maximal (50%) stimulation) were calculated.For that purpose the software program GraphPad PRISM, version 3.0(GraphPad software Inc., San Diego) was used.

[0432] In a similar way FSH agonistic activity of compounds were testedin CHO cells transfected with the luciferase reporter gene and the humanFSH receptor. Results are shown in Table 1.

[0433] In vivo bioactivity

[0434] To measure in vivo activity of LH/FSH receptor agonisticcompounds ovulation induction in immature mice were studied. In thisassay immature female mice were primed with urinary FSH (Humegon 12.5IU/animal). Approximately 48 hours later the animals were treated with aLH/FSH agonistic compound, the preparation of which is described inexamples 1, 4, 9 and 17, at a dose-level of 50 mg/kg. The animals werekilled 24 hours after LH/FSH agonist treatment and the number of ova inthe oviduct was microscopically assessed. On average 10-15 animals weretested. The mean number of ova amounted 8 with the exception of thecompound of example 17 (which was 0.4). TABLE 1 name example LH EC50 (M)FSH EC50 (M) tert-Butyl 5-amino-2- 1 2.73E−09 5.69E−08methylthio-4-(3-((N-methyl-N-(2- hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide tert-Butyl5-amino-2-methylthio-4-(3-((N- 2 5.42E−09 8.06E−07(1-hydroxy-2-methyl-prop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-methylthio-4-(3-((N- 3 5.97E−09 1.44E−06(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-methylthio-4-(3-((N- 4 7.00E−09 1.01E−07ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-methylthio-4-(3-((N- 5 9.63E−09 3.48E−07(R-1-methoxycarbonyl-2-methyl-prop-1-yl)-glycinyl)-amino)-phenyl)-thieno[2,3- d]pyrimidine-6-carboxamidetert-Butyl 5-amino-2-methylthio-4-(3- 6 1.00E−08 1.18E−06((N,N-bis-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-methylthio-4-(3- 7 1.22E−08 1.13E−06((2,3-dihydroxy-prop-1-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-methylthio-4-(3- 8 2.64E−08 1.00E−06((1,3-dihydroxyprop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-phenyl-4-(3-((N- 9 4.82E−09 6.06E−07ethyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-phenyl-4-(3-(N- 10 1.01E−08 2.48E−06(methoxycarbonylmethyl)-gycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(2-furyl)-4-(3-((N- 11 4.00E−09 6.16E−07methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(2-furyl)-4-(3-(N-(1- 12 1.18E−08 2.97E−06hydroxy-2-methyl-prop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(2-furyl)-4-(3-(N- 13 1.21E−08 2.55E−06(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(2-thienyl)-4-(3-((N- 14 5.28E−09 1.44E−06methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(2-thienyl)-4-(3-((N- 15 1.82E−08 3.21E−06(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(2-thienyl)-4-(3- 16 2.13E−08 6.81E−06((N,N-di-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-ethylamino-4-(3- 17 1.04E−08 1.25E−06((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(N,N- 18 1.45E−08 1.63E−06dimethylamino)-4-(3-((N-methyl-N-(2-hydroxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide tert-Butyl5-amino-2-ethylamino-4-(3- 19 2.29E−08 2.30E−06((N-(methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-isopropylamino-4- 20 4.24E−08 2.95E−06(3-((N-ethyl-N-(2-methoxyethyl)- glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide tert-Butyl 5-amino-2-allylamino-4-(3-((N- 215.70E−08 5.60E−07 (methoxycarbonylmethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-methoxy-4-(3-((N- 22 1.16E−08 9.53E−07ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-allyloxy-4-(3-((N- 23 3.68E−08 1.74E−06ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-isopropoxy-4-(3- 24 7.82E−08 2.51E−06((N-ethyl-N-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(4-pyridyl)-4-(3-((N- 25 3.47E−08 4.00E−07(1-hydroxy-2-methyl-prop-2-yl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxamide tert-Butyl5-amino-2-(4-pyridyl)-4-(3- 26 3.82E−08 1.23E−06((N,N-bis-(2-methoxyethyl)-glycinyl)-amino)-phenyl)-thieno[2,3-d]pyrimidine-6- carboxamide

1. A thieno[2,3-d]pyrimidine derivative according to general formula I,

or a pharmaceutically acceptable salt thereof, wherein X is O or H,H Ais S, NH, N(R⁶), O or a bond; R¹ is (1-4c)alkyl, (2-4c)alkenyl, phenylor (2-5c)heteroaryl, the phenyl or heteroaryl ring optionally beingsubstituted with one or more of the group of substituents: hydroxy,halogen, nitro, trifluoromethyl, cyano, amino or (1-4c)(di)alkylamino:R² is H, (1-4C)alkyl, (1-4C)alkoxy(2-4C)alkyl or hydroxy(2-4C)alkyl; R³and R⁴ can be independently selected from H, (1-4C)alkyl andhydroxy(1-4C)alkyl; is R⁵ is H or (1-4C)alkyl, and R⁶ can be selectedfrom the same groups as described for R¹.
 2. The compound according toclaim 1 wherein X is H,H.
 3. The compound according to claims 1 or 2wherein R⁵ is (1-4C)alkyl.
 4. The compound according to claims 1-3wherein R³=R⁴.
 5. The compound according to claims 1-4 wherein R³=R⁴=H.6. The compound according to claims 1-5 wherein R² is (1-4C)alkyl. 7.The compound according to claims 1-6 for use in therapy.
 8. Apharmaceutical composition comprising a thieno[2,3-d]pyrimidine compoundaccording to claims 1-6 or a pharmaceutically acceptable salt or solvatethereof, in admixture with a pharmaceutically acceptable auxiliary. 9.Use of thieno[2,3-d]pyrimidine compounds according to claims 1-6 or apharmaceutically acceptable salt or solvate thereof for the manufactureof a medicament for the control of fertility.
 10. A method to treatfertility disorders in patients in need thereof by administration of aneffective amount of a compound according to claims 1-6.